Summary of Current Projects: [unreadable] [unreadable] (1) Magnetic Resonance Imaging (MRI) of Drug Movement in the Eye: A number of inflammatory and neoplastic diseases of the eye are currently treated by repeated intravitreal drug injection. Transscleral delivery has emerged as a more attractive method for treating retinal disorders, because it offers localized delivery of drugs as a less invasive method compared to intravitreal administration. Numerous novel transscleral drug delivery systems, ranging from microparticles to implants, have been reported. However, transscleral delivery is currently not as clinically effective as intravitreal delivery in the treatment of retinal diseases, and this lack of success must be evaluated. Transscleral drug delivery systems require drugs to permeate through several layers of ocular tissue (sclera, choroid-Bruchs membrane, retinal pigment epithelium) to reach the neuroretina. In addition, clearance of administered drugs occurs by uptake into the conjunctival and choroidal blood and possibly into lymph. This study is aimed at gaining a better understanding of these clearance mechanisms so that better delivery methods may be designed. In this project, we are using MRI as a non-invasive, in vivo method to track the movement of small and large molecule drug surrogates in the form, respectively, of the MRI-tracers Gd-DPTA and Gd-albumin. The delivery of these agents is by bolus injection or by slow continuous infusion either into the subconjunctival or intra-scleral space of the eyes of anesthetized rabbits. The drug levels in an eye are monitored locally with MRI for up to eight hours. Finite element mathematical models, which incorporate the physico-chemical properties of the drug and the physiology of the eye, are used to interpret the MRI data and to gain a better understanding of the drug transport processes in the eye. These models can be useful in guiding the design of optimal drug therapy methodologies. [unreadable] [unreadable] (2) Sustained-Release Devices for Intraocular Drug Delivery: We are investigating the use of sustained drug release devices that could release drugs into the body for periods as long as months. We are currently testing the use of a thermosetting polymer, ReGel (Protherics, Inc.), as an injectable agent for long term release of drugs into the eye. This technique would eliminate the need for frequent invasive bolus liquid injections. Our experiments include subconjunctival injections of ReGel containing fluorescently labeled ovalbumin into rat eyes and Gd-albumin into rabbit eyes. In the rat experiments, a drug marker, in the form of fluorescently-labelled ovalbumin (Alexafluor-ova), is incorporated into a ReGel solution, which is liquid at room temperature, and is then injected into the subconjunctival space of rats, where it forms a slow releasing gel at 37 centigrade. Animals are sacrificed at serial time points from one hour up to four weeks and enucleated eyes are examined in histological sections by fluorescent microscopy and by tissue extraction for the quantitative assessment of fluorescent label. In comparable experiments in rabbits, ReGel loaded with Gd-albumin is injected into the subconjunctival space. MRI images are obtained over time to track the movement of this surrogate drug and to determine the residence time of the Gadolinium labeled albumin in the subconjunctival depot. If sustained levels of the drug marker are achieved in the neuroretina for periods up to four weeks or more, then the ReGel system could become a candidate for clinical applications of long term drug delivery to the eye. [unreadable] [unreadable] (3) Influence of Proteoglycans on Axonal Growth: Chondroitin sulfate proteoglycans (CSPGs) are major components of glial scars that form soon after spinal chord injury. It is believed that CSPGs significantly inhibit axonal growth and regeneration. CSPGs consist of a protein core with large glycosaminoglycan (GAG) sugar side chains. Removal of the GAG chains by chondroitinases has been shown to restore neuronal growth activity. We are seeking to determine which of the specific GAG sugars, chondroitin sulfate A or chondroitin sulfate C, is responsible for the neuronal inhibition. We are also planning to study potential sustained release devices that can release the chondroitinases into the injury sites to reduce the presence of the GAG sugars on the proteoglycans and thus encourage axonal regeneration. The project proposes a series of in vitro experiments to study the effects of the specific sugars on the growth and spreading of neuronal PC-12 cells into a collagen matrix in the presence and absence of particular sugars. Cell growth will be monitored by microscopy. In addition, we will test the release, distribution, and effectiveness of chondroitinase enzyme delivered from nanoparticles into an in vitro gel matrix. Subsequent experiments would involve the delivery of the nano-encapsulated enzyme into rats with spinal chord injury to investigate the effectiveness of this method on nerve regeneration in vivo.